Project Summary/Abstract: The current standard-of-care for stage IIB-IVA cervical cancer includes the chemotherapeuticdrug,cisplatin,inconjunctionwithlocalradiotherapy.However,thefive-yearsurvivalinmost stage IIB-IVA cervical cancer patients is ~30%, suggesting that an additional treatment strategy (e.g. immunotherapy)isneededtoimprovepatientoutcomes.Werecentlyfoundthatchemotherapywithcisplatinor radiotherapyinduceatransientaccumulationofdendriticcellsintumorlocithatcreatesawindow-of-opportunity wherein intratumoral (i.t.) protein/peptide antigen administration can effectively prime and expand local tumor antigen-specificCD8+Tcells,leadingtosynergistictherapeuticantitumoreffects.Furthermore,thiscombination treatmentleadstothepresentationofantigenicpeptidebyimmunosuppressivestromalcellsofthetumor,which are thus rendered susceptible to antigen-specific CD8+ T cell-mediated killing. Together, this results in potent antigen-specific CD8+ T cell immune responses and antitumor effects. TA-CIN is a tandem fusion protein composed of HPV16 L2-E7-E6 and is administered as a filterable aggregrate to promote uptake by antigen- presentingcells.Becausecervicalcancerisaccessibletoinjectionandconcurrentchemoradiationservesasthe standardcareforstageIIB-IVAcervicalcancer,wewillvaccinatestageIIB-IVAcervicalcancerpatientswithTA- CIN i.t. during their chemoradiotherapy. Several clinical trials indicate that TA-CIN has an outstanding safety profile. With the recent approval of an anti-PD-1 antibody, Pembrolizumab, to treat advanced cervical cancer, we will evaluate i.t. TA-CIN vaccination with concomitant immune checkpoint blockade therapy to achieve enhanced immune and therapeutic responses in a novel spontaneous cervicovaginal carcinoma preclinical model. The Specific Aims of this study are to (1) evaluate the safety of TA-CIN i.t. administration during chemoradiation in patients with stage IIB-IVA HPV16(+) or HPV16(?) cervical cancer;? (2) characterize HPV16 E6/E7-specific T cell-mediated and L2-specific humoral immune responses in stage IIB-IVA cervical cancer patients i.t. vaccinated with TA-CIN;? (3) determine the subset population of immune cells infiltrating the tumor bed, local expression of PD-1 and PD-L1, and apoptotic tumor cell death in HPV16-associated stage IIB-IVA cervical cancer patients receiving i.t. TA-CIN vaccination;? and (4) characterize the HPV16(+) antigen-specific CD8+ T cell-mediated immune responses and therapeutic antitumor effects in a spontaneous HPV16 E6/E7- expressing cervicovaginal carcinoma model treated with i.t. TA-CIN vaccination with or without checkpoint blockade treatment. In sum, this proposal exploits the temporally-permissive tumor microenvironment induced bychemoradiationandtheavailabilityofclinical-gradeTA-CINtopotentiallyenhanceHPV-specificimmunityand antitumor effects in stage IIB-IVA cervical cancer patients. This may lead to a safe new immunotherapy to complementchemoradiation,whichnotonlybenefitscervicalcancerpatients,butmayalsobeeffectiveagainst otherHPV-associatedmalignancies.